Anion exchange resin adsorbed fraction immunopotentiator and promoter for recovery from loaded stress originating in matsutake mushroom

ABSTRACT

An adsorption fraction of a liquid mixture by an anion exchange resin, wherein the liquid mixture is obtainable by mixing a hot water extract of a mycelium of  Tricholoma matsutake  FERM BP-7304 with an alkaline solution extract of a mycelial residue obtained when preparing the hot water extract, is disclosed. Further, an immune enhancing agent and an agent for promoting a recovery from stress comprising the above-described novel adsorption fraction as an active ingredient, are disclosed.

TECHNICAL FIELD

The present invention relates to a novel fraction adsorbed by an anionexchange resin from extracts of Tricholoma matsutake, an immuneenhancing agent and an agent for promoting a recovery from stresscontaining the same. The immune enhancing agent or agent for promoting arecovery from stress according to the present invention may beadministered as a medicament or in various forms, for example, eatableor drinkable products such as health foods (preferably functional foods)or feeds. The term “foods” as used herein include drinks. Further, theagent of the present invention may be administered in the form of anoral hygienic composition which is temporarily kept in the mouth butthen spat out, retaining almost none of the components, for example, adentifrice, a mouthwash agent, a chewing gum, or a collutorium, or inthe form of an inhalation drawn in through the nose.

BACKGROUND ART

The present inventors demonstrated that an administration of biologicalresponse modifiers (BRM) into a primary tumor alleviated not only theprimary tumor but also a metastatic tumor, by a “double grafted tumorsystem” in mice developed by part of the present inventors andcollaborators (Ebina, Biotherapy, 14, 379-394, 2000; Ebina et al., Ganto Kagaku-ryouhou (Jpn J. Cancer & Chemotherapy), 18, 1812-1816, 1991;and Ebina et al., Biotherapy, 12, 495-500, 1998). A mere primary tumorcan be extirpated by an operation. However, in cases of metastatic foci(particularly small metastatic foci not detectable with theexamination), if a primary tumor is extirpated, the metastatic focibegins to proliferate, and thus the treatment cannot cure cancer. Inother words, an important problem in cancer treatment is the treatmentof metastatic foci.

Hitherto, it was believed that a local administration of an agent tocancer was not preferred because of the spread of tumor cells. However,the present inventors found that not only a primary focus but alsometastatic foci was regressed by local administration of a BRM to cancer(Ebina et al., Jpn.J.Cancer Res., 77, 1034-1042, 1986). In the study, aprotein-bound polysaccharide PSK, extracted from mycelia of Coriolusversicolor (FR.) Quél., was able to cure not only a primary tumor butalso metastatic foci, whereas lentinan, β-glucan purified from fruitbodies of Lentinus edodes, did not exhibit an antitumor activity.Further, cepharanthin, extracted from Stephania cepharantha Hayata,exhibited an antitumor activity to right and left tumors [Ebina et al.,Gan to Kagaku-ryouhou (Jpn J. Cancer & Chemotherapy), 28, 211-215,2001], but the purified alkaloid exhibited a weak antitumor activity.

It was found from the above results that, among BRMs, purifiedsubstances obtained by Western medicine exhibited a weak or littleantitumor activity, whereas extracts containing an active ingredientwere superior thereto [Ebina et al., Gan to Kagaku-ryouhou (Jpn J.Cancer & Chemotherapy), 28, 1515-1518, 2001]. Further, a new concept ofa third medicine or integrated medicine, which was different from amixture of several kinds of extracts, such as Chinese drugs in Chinesemedicine, was formulated [Ebina et al., Igaku no Ayumi (Journal ofClinical and Experimental Medicine), 194, 690-692, 2000].

Further, it is known that Tricholoma matsutake (S.Ito & Imai) Sing.contains many physiologically active substances. For example, JapaneseExamined Patent Publication (Kokoku) No. 57-1230 and Japanese Patent No.2767521 disclose various antitumor substances contained in Tricholomamatsutake. The above Japanese Examined Patent Publication (Kokoku) No.57-1230 discloses that emitanine-5-A, emitanine-5-B, emitanine-5-C, andemitanine-5-D, which are separated and purified from a liquid extractobtained by extracting a broth of Tricholoma matsutake mycelia with hotwater or a diluted alkaline solution, exhibits an activity of inhibitinga proliferation of sarcoma 180 cells. The above Japanese Patent No.2767521 discloses that a protein with a molecular weight of 0.2 to 0.21million (a molecular weight of a subunit=0.1 to 0.11 million) which isseparated and purified from an extract of Tricholoma matsutake fruitbodies with water exhibits an antitumor activity.

Further, the present inventors found that a hot water extract ofTricholoma matsutake or an alkaline solution extract of Tricholomamatsutake, or an adsorption fraction of a hot water extract ofTricholoma matsutake or an alkaline solution extract of Tricholomamatsutake by an anion exchange resin exhibits an immuno-enhancingactivity (WO01/49308)

The present inventors made an intensive search for another substancehaving an immuno-enhancing activity, and as a result, newly found that apartially purified fraction derived from mycelia obtained by culturingthe specific Tricholoma matsutake strain by the specific culturingmethod exhibited such an immuno-enhancing activity. Further, the presentinventors found that the fraction was different from known fractions,such as the adsorption fraction by an anion exchange disclosed inWO01/49308, with respect to physicochemical properties, and that thefraction exhibited an activity of promoting a recovery from stress. Thispresent invention is based on the above findings.

DISCLOSURE OF THE INVENTION

Therefore, the object of the present invention is to provide a novelfraction adsorbed (hereinafter referred to as adsorption fraction) by ananion exchange resin from Tricholoma matsutake, a novel immune enhancingagent, and a novel agent for promoting a recovery from stress.

The above object can be solved by the present invention, i.e., anadsorption fraction of a liquid mixture by an anion exchange resin,wherein the liquid mixture is obtainable by mixing a hot water extractof a mycelium of Tricholoma matsutake FERM BP-7304 with an alkalinesolution extract of a mycelial residue obtained when preparing the hotwater extract, and

-   -   (a) the content of carbohydrates in the adsorption fraction is        60 to 72% as a glucose equivalent determined by a        phenol-sulfuric acid method, and    -   (b) the content of proteins in the adsorption fraction is 28 to        40% as an albumin equivalent determined by a copper-Folin        method.

Further, the present invention relates to an immune enhancing agentcomprising the above-mentioned adsorption fraction as an activeingredient.

The present invention relates to an immune enhancing compositioncomprising the adsorption fraction and a pharmaceutically acceptablecarrier.

The present invention relates to an immune enhancing health foodcomprising the adsorption fraction alone or, optionally, with one ormore food components.

According to a preferred embodiment of the immune enhancing health foodof the present invention, the health food is a functional food.

The present invention relates to a method for an immune enhancement,comprising administering to a subject in need thereof the adsorptionfraction in an amount effective therefor.

The present invention relates to use of the adsorption fraction in themanufacture of an immune enhancing composition or health food.

Further, the present invention relates to an agent for treating orpreventing metastatic foci, comprising the adsorption fraction as anactive ingredient.

The present invention relates to a composition for treating orpreventing metastatic foci, comprising the adsorption fraction and apharmaceutically acceptable carrier.

The present invention relates to a health food for treating orpreventing metastatic foci, comprising the adsorption fraction alone or,optionally, with one or more food components.

According to a preferred embodiment of the health food for treating orpreventing metastatic foci of the present invention, the health food isa functional food.

The present invention relates to a method for treating or preventingmetastatic foci, comprising administering to a subject in need thereofthe adsorption fraction in an amount effective therefor.

The present invention relates to use of the adsorption fraction in themanufacture of a composition or health food for treating or preventingmetastatic foci.

Further, the present invention relates to an agent for increasing aserum Immunosuppressive Acidic Protein (IAP) value, comprising theadsorption fraction as an active ingredient.

The present invention relates to a composition for increasing a serumIAP value, comprising the adsorption fraction and a pharmaceuticallyacceptable carrier.

The present invention relates to a health food for increasing a serumIAP value, comprising the adsorption fraction alone or, optionally, withone or more food components.

According to a preferred embodiment of the health food for increasing aserum IAP value of the present invention, the health food is afunctional food.

The present invention relates to a method for increasing a serum IAPvalue, comprising administering to a subject in need thereof theadsorption fraction in an amount effective therefor.

The present invention relates to use of the adsorption fraction in themanufacture of a composition or health food for increasing a serum IAPvalue.

Further, the present invention relates to an agent for promoting arecovery from stress, comprising the adsorption fraction as an activeingredient.

The present invention relates to a composition for promoting a recoveryfrom stress, comprising the adsorption fraction and a pharmaceuticallyacceptable carrier.

The present invention relates to a health food for promoting a recoveryfrom stress, comprising the adsorption fraction alone or, optionally,with one or more food components.

According to a preferred embodiment of the health food for promoting arecovery from stress of the present invention, the health food is afunctional food.

The present invention relates to a method for promoting a recovery fromstress, comprising administering to a subject in need thereof theadsorption fraction in an amount effective therefor.

The present invention relates to use of the adsorption fraction in themanufacture of a composition or health food for promoting a recoveryfrom stress.

Further, the present invention relates to an immune enhancing agentcomprising the adsorption fraction as an active ingredient, with theproviso that an agent for treating or preventing metastatic foci and anagent for increasing a serum IAP value are excluded from the immuneenhancing agent.

The present invention relates to an immune enhancing compositioncomprising the adsorption fraction and a pharmaceutically acceptablecarrier, with the proviso that a composition for treating or preventingmetastatic foci and a composition for increasing a serum IAP value areexcluded from the immune enhancing composition.

The present invention relates to an immune enhancing health foodcomprising the adsorption fraction alone or, optionally, with one ormore food components, with the proviso that a health food for treatingor preventing metastatic foci and a health food for increasing a serumIAP value are excluded from the immune enhancing health food.

According to a preferred embodiment of the immune enhancing health foodof the present invention, the health food is a functional food.

The present invention relates to a method for an immune enhancement,comprising administering to a subject in need thereof the adsorptionfraction in an amount effective therefor, with the proviso that a methodfor treating or preventing metastatic foci and a method for increasing aserum IAP value are excluded from the method for an immune enhancement.

The present invention relates to use of the adsorption fraction in themanufacture of an immune enhancing composition or health food, with theproviso that a composition or health food for treating or preventingmetastatic foci and a composition or health food for increasing a serumIAP value are excluded from the immune enhancing composition or healthfood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a time course for a tumor diameter(mean±standard deviation) of the right tumor in a double grafted tumorsystem.

FIG. 2 is a graph showing a time course for a tumor diameter(mean±standard deviation) of the left tumor in a double grafted tumorsystem.

FIG. 3 is a time course for a serum IAP value after administering the M1fraction or M2 fraction.

FIG. 4 is a graph showing that a gene expression of IL-12 in mouse cellsfrom a mesenterium lymph node was induced by orally administering the M2fraction to mice.

FIG. 5 is a graph showing extension of a survival time and effects ofanti-IL-12 antibody treatment in mice in which a tumor (P815 tumorcells) was immunized and the M2 fraction was orally administered.

FIG. 6 is a graph showing extension of a survival time and effects ofanti-IL-12 antibody treatment in mice in which a tumor (colon26 tumorcells) was immunized and the M2 fraction was orally administered

FIG. 7 is a spectrum obtained by a ¹H one-dimensional NMR measurement ofthe adsorption fraction M2.

FIG. 8 is a spectrum obtained by a ¹³C one-dimensional NMR measurementof the adsorption fraction M2.

FIG. 9 is a broad spectrum obtained by a ¹³C one-dimensional NMRmeasurement of the adsorption fraction M2.

FIG. 10 is a CD spectrum obtained by a circular dichroism analysis ofthe adsorption fraction M2.

FIG. 11 is a spectrum obtained by an infrared spectroscopic analysis ofthe adsorption fraction M2.

FIG. 12 is a spectrum obtained by an ultraviolet spectroscopic analysisof the adsorption fraction M2.

FIG. 13 is a spectrum obtained by an ESR analysis of the adsorptionfraction M2.

FIG. 14 is a broad spectrum obtained by an ESR analysis of theadsorption fraction M2.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

The immune enhancing agent or agent for promoting a recovery from stressof the present invention contains as an active ingredient “an adsorptionfraction of a liquid mixture by an anion exchange resin”, the liquidmixture being obtained by mixing “a hot water extract of a mycelium ofTricholoma matsutake FERM BP-7304 [Tricholoma matsutake (S.Ito & Imai)Sing. CM6271]” (hereinafter sometimes referred to as “hot water extractof the mycelium” ) with “an alkaline solution extract of a mycelialresidue obtained when preparing the hot water extract of the mycelium”(hereinafter sometimes referred to as “alkaline solution extract of themycelial residue”), wherein

-   -   (a) the content of carbohydrates in the adsorption fraction is        60 to 72% (preferably 62 to 70%) as a glucose equivalent        determined by a phenol-sulfuric acid method, and    -   (b) the content of proteins in the adsorption fraction is 28 to        40% (preferably 30 to 38%) as an albumin equivalent determined        by a copper-Folin method. Hereinafter, the above “liquid        mixture” obtainable by mixing the hot water extract of the        mycelium with the alkaline solution extract of the mycelial        residue is sometimes referred to as “extract mixture”.

The adsorption fraction of the extract mixture by an anion exchangeresin, which is used as the active ingredient in the immune enhancingagent or agent for promoting a recovery from stress of the presentinvention, may be prepared by, for example, but is by no means limitedto, a process comprising the steps of:

-   -   culturing the Tricholoma matsutake FERM BP-7304 strain by a tank        culture to obtain the mycelium thereof (hereinafter referred to        as a culturing step);    -   extracting the resulting mycelium with hot water to obtain the        hot water extract of the mycelium (hereinafter referred to as a        hot water extracting step);    -   extracting the remaining mycelial residue, after the extraction        with hot water, with an alkaline solution to obtain the alkaline        solution extract of the mycelial residue (hereinafter referred        to as an alkaline solution extracting step);    -   mixing the hot water extract of the mycelium and the alkaline        solution extract of the mycelial residue to obtain the extract        mixture, and adsorbing the resulting extract mixture by an anion        exchange resin (hereinafter referred to as an anion exchange        resin-adsorbing step); and    -   eluting the adsorption fraction with an appropriate eluting        solution (hereinafter referred to as an eluting step).

The Tricholoma matsutake strain FERM BP-7304 (International ApplicationPCT/JP01/08876) used in the culturing step was deposited in theInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology [(Former Name) National Institute ofBioscience and Human-Technology Agency of Industrial Science andTechnology (Address: AIST Tsukuba Central 6, 1-1, Higashi 1-chomeTukuba-shi, Ibaraki-ken 305-8566 Japan)] on Sep. 14, 2000. TheTricholoma matsutake strain FERM BP-7304 was established as a strain byculturing a piece of fruit body from a Tricholoma matsutake strainCM6271 collected in Kameoka-shi, Kyoto, Japan and then culturing it invitro, and is maintained in the Biomedical Research Laboratories, KurehaChemical Industry Co. Ltd.

The macroscopical observation of a fruit body of the Tricholomamatsutake strain FERM BP-7304 is identical with that of a Tricholomamatsutake fruit body described in Rokuya Imaseki and Tsuguo Hongo,“Gensyoku Nihon Shinkinrui Zukan (Colored Illustrations of Mushrooms ofJapan) (I)”, Hoiku-sha (Osaka, Japan), 1957, plate 9 and page 26. TheTricholoma matsutake strain FERM BP-7304 can be subcultivated ormaintained on a MATSUTAKE medium. A large-scale cultivation of myceliaof the Tricholoma matsutake strain FERM BP-7304 can be carried out byinoculating the strain into a liquid medium and then performing, forexample, a static culture, a shake culture, or a tank culture.

When mycelia of the Tricholoma matsutake strain FERM BP-7304 areinoculated on a MATSUTAKE medium, white hyphae grow radially andthickly, and form large colonies. According to an observation by ascanning electron microscope, a large number of branched mycelia havinga diameter of 1 to 2 μm exist and projections having a height ofapproximately a few μm are observed at the side of mycelia. In thisconnection, the Tricholoma matsutake strain FERM BP-7304 can besubcultured or cultured, generally in the form of mycelia, but sometimesin the form of fruit bodies.

Mycological features of the Tricholoma matsutake strain FERM BP-7304will be explained hereinafter.

(1) Cultural and Morphological Features on Malt Extract Agar Medium

When the Tricholoma matsutake strain FERM BP-7304 is inoculated on amalt extract agar medium, white hyphae grow radially and thickly, andform colonies. A diameter of the colony after 30 days from theinoculation is approximately 4 cm.

(2) Cultural and Morphological Features on Czapek's Agar Medium, OatmealAgar Medium, Synthetic Mucor Agar Medium, and Medium for Assaying PhenolOxidase Reaction

When the Tricholoma matsutake strain FERM BP-7304 is inoculated on aCzapek's agar medium, an oatmeal agar medium, a synthetic mucor agarmedium, or a medium for assaying a phenol oxidase reaction, a growth ofhyphae is not observed after a month from the inoculation.

(3) Cultural and Morphological Features on YpSs Agar Medium

The Tricholoma matsutake strain FERM BP-7304 grows as a mat-like myceliahaving a white gloss on a YpSs agar medium. A growth area after 30 daysfrom the inoculation is approximately 5 mm in radius.

(4) Cultural and Morphological Features on Glucose and Dry Yeast AgarMedium

The Tricholoma matsutake strain FERM BP-7304 grows as a mat-like myceliahaving white gloss on a glucose and dry yeast agar medium. A growthdistance after 30 days from the inoculation is approximately 2 mm.

(5) Optimal Growth Temperature and Range of Temperature

After 100 mL-conical flasks each containing 10 mL of sterile medium (3%glucose and 0.3% yeast extract, pH 7.0) were inoculated withapproximately 2 mg of a piece of the Tricholoma matsutake strain FERMBP-7304 mycelia of the present invention, cultivation was performed atvarious temperatures from 5 to 35° C. After cultivation for 28 days,mycelia were taken from the flasks, washed thoroughly with distilledwater, and dried, and then each weight of mycelia was measured. As aresult, it was found that the weight of mycelia increased linearly at arange of 5 to 15° C., and gently at a range of 15 to 25° C. Mycelia didnot grow at 27.5° C. or more. The optimal growth temperature is 15 to25° C.

(6) Optimal Growth pH and Range of pH

A growth pH value was examined by preparing various media having a pH ina range of 3.0 to 8.0. These media were prepared by adjusting a pH of aliquid medium (3% glucose and 0.3% yeast extract) with 1 mol/L HCL or 1mol/L potassium hydroxide. Each medium was sterilized through a filter,and then 10 mL of the sterile medium was poured into a 100 mL-conicalflask (previously sterilized). After approximately 2 mg of a piece ofthe Tricholoma matsutake strain FERM BP-7304 mycelia was inoculated,cultivation was performed at 22° C. After cultivation for 28 days,mycelia were taken from the flasks, washed thoroughly with distilledwater, and dried, and then each weight of mycelia was measured. As aresult, the limit for growth of mycelia was in a range of a pH 3.0 to7.0. The optimal growth pH was 4.0 to 6.0.

(7) Formation of Confront Line by Confrontation Culture

A block (approximately 3 mm×3 mm×3 mm) of the Tricholoma matsutakestrain FERM BP-7304 and each block (approximately 3 mm×3 mm×3 mm) of 13kinds of known Tricholoma matsutake strains were inoculated with a spaceof approximately 2 cm therebetween on a MATSUTAKE medium. Aftercultivation was carried out at 22° C. for 3 weeks, it was observedwhether or not a zone appeared at the boundary between two colonies.

As a result, it was found that the Tricholoma matsutake strain FERMBP-7304 did not form a clear zone with respect to the 13 kinds of knownTricholoma matsutake strains. In this connection, it is considered thatTricholoma matsutake does not form a zone in a confrontation culture.Among the 13 kinds of known Tricholoma matsutake strains, nocombinations formed a clear zone.

(8) Auxotrophy

After a 100 mL-conical flask containing 10 mL of a sterile syntheticmedium for mycorrhizae (Ohta et al., Trans. Mycol. Soc. Jpn., 31, .323,1990) was inoculated with approximately 2 mg of a piece of theTricholoma matsutake strain FERM BP-7304 mycelia of the presentinvention, cultivation was performed at 22° C. After cultivation for 42days, the mycelia were taken from the flask, washed thoroughly withdistilled water, and dried, and then the weight of mycelia was measuredto obtain 441 mg of mycelia.

Each medium in which one of 28 kinds of sugar-related substances wassubstituted for glucose as a carbon (C) source in the synthetic mediumfor mycorrhizae was inoculated with the Tricholoma matsutake strain FERMBP-7304 of the present invention, and cultivation was performed. Afterthe cultivation, each weight of mycelia was measured.

As a result, an order of the sugar-related substances from that whichachieved the heaviest weight to that which achieved the lightest weightwas as follows:

-   -   wheat starch>corn        starch>dextrin>methyl-β-glucoside>cellobiose>mannose>fructose>arabinose>sorbitol>glucose>lactose>glycogen>mannitol>ribose>maltose>trehalose>galactose>raffinose>melibiose>N-acetylglucosamine.

In this connection, mycelia did not grow in each medium containingcellulose, dulcitol, sucrose, xylose, methyl-α-glucoside, inulin,inositol, or sorbose.

Further, each medium in which one of 15 kinds of nitrogen-relatedsubstances was substituted for ammonium tartrate as a nitrogen (N)source in the synthetic medium for mycorrhizae was inoculated with theTricholoma matsutake strain FERM BP-7304 of the present invention, andcultivation was performed. After the cultivation, each weight of myceliawas measured.

As a result, an order of the nitrogen-related substances from that whichachieved the heaviest weight to that which achieved the lightest weightwas as follows:

-   -   corn steep liquor>soybean peptone>milk peptone>ammonium        nitrate>ammonium sulfate>ammonium tartrate>ammonium        carbonate>asparagine>ammonium phosphate>ammonium chloride>sodium        nitrate>meat extract>yeast extract>casamino        acids>chlorella>tryptone>potassium nitrate.

Furthermore, each medium in which one component among minerals andvitamins in the above synthetic medium was removed was inoculated withthe Tricholoma matsutake strain FERM BP-7304 of the present invention,and cultivation was performed. After the cultivation, each weight ofmycelia was measured.

As a result, a deficiency of any one of calcium chloride dihydrate,manganese sulfate (II) pentahydrate, zinc sulfate heptahydrate, cobaltsulfate heptahydrate, copper sulphate pentahydrate, nickel sulfatehexahydrate, thiamin hydrochloride, nicotinic acid, folic acid, biotin,pyridoxine hydrochloride, carnitine chloride, adenine sulfate dihydrate,or choline hydrochloride did not affect the weight of mycelia. Incontrast, when any one of magnesium sulfate heptahydrate, iron chloride(II), or sodium dihydrogen phosphate was removed, the weight of myceliawas remarkably lowered. From these results, it is considered thatmagnesium, iron, phosphorus, and potassium are essential for the growthof the Tricholoma matsutake strain FERM BP-7304 of the presentinvention.

(9) Base Composition of DNA (GC Content)

The GC content of the Tricholoma matsutake strain FERM BP-7304 is 49.9%.

(10) DNA Patterns Generated by a RAPD Method

With respect to each DNA pattern generated by a RAPD (random amplifiedpolymorphic DNA) method using any one of six kinds of primers (10 mer)for PCR (polymerase chain reaction), the Tricholoma matsutake strainFERM BP-7304 was compared with 44 kinds of known Tricholoma matsutakestrains (for example, IFO 6915 strain; Institute for Fermentation,Osaka). As a result, it was confirmed that DNA patterns of theTricholoma matsutake strain FERM BP-7304 differed from those of 44 kindsof Tricholoma matsutake strains.

As a medium used in the culturing step, for example, a medium composedof glucose and yeast extract may be used.

The concentration of glucose contained in the medium is preferably 0.01%to 15%, more preferably 1% to 10%, most preferably 3%. The concentrationof yeast extract contained in the medium is preferably 0.01% to 3%, morepreferably 0.1% to 0.6%, most preferably 0.3%. The medium is preferablypH 2.5 to 8, more preferably pH 4 to 7, most preferably pH 6.

The culturing temperature in the culturing step is preferably 10 to 26°C., more preferably 15 to 25° C., most preferably 25° C.

The period for cultivation is preferably 1 to 20 weeks, more preferably2 to 10 weeks, most preferably 10 weeks.

As a method for separating mycelia from a broth obtained by cultivation,for example, filtration (for example, filtration with a filter cloth) orcentrifugation may be used. It is preferred to wash the resultingmycelia with, for example, distilled water, before the next hot waterextracting step is carried out. Further, it is preferred to crush,grind, or pulverize mycelia, to enhance the extraction efficiency.

The temperature of hot water used in the hot water extracting step ispreferably 60 to 100° C., more preferably 80 to 98° C. It is preferredto carry out the extraction with stirring or shaking, so that theextraction efficiency is enhanced. An extracting time may beappropriately determined in accordance with, for example, the form ofmycelia (for example, a crushed, ground, or pulverized form, iftreated), the temperature of hot water, or with or without stirring orshaking or a treating condition thereof, but is generally 1 to 6 hours,preferably 2 to 3 hours.

After the hot water extraction, a hot water extract of mycelia can beseparated from a mycelial residue by an appropriate separating method,such as centrifugation or filtration.

An alkaline solution used in the alkaline solution extracting step maybe, for example, but is by no means limited to, an aqueous solution of ahydroxide of an alkaline metal (such as sodium or potassium),particularly sodium hydroxide. The alkaline solution is preferably pH 8to 13, more preferably pH 9 to 12. The alkaline solution extraction maybe carried out preferably at 0 to 30° C., more preferably at 0 to 25° C.An extraction time may be appropriately determined in accordance with,for example, the form of a mycelial residue (for example, a crushed,ground, or pulverized form, if treated), the temperature or pH of analkaline solution, or with or without stirring or shaking or a treatingcondition thereof, but is generally 30 minutes to 5 hours, preferably 1to 3 hours.

After the alkaline solution extraction, an alkaline solution extract ofthe mycelial residue can be separated from a mycelial residue by anappropriate separating method, such as centrifugation or filtration.

It is preferred to neutralize the resulting alkaline solution extract ofthe mycelial residue, before the next anion exchange resin-adsorbingstep is carried out.

The extract mixture obtained by mixing the hot water extract of myceliaprepared in the hot water extracting step with the alkaline solutionextract of the mycelial residue prepared in the alkaline solutionextracting step may be used, without a further treatment (i.e., togetherwith insolubles), in the next anion exchange resin-adsorbing step.However, it is preferred to remove the insolubes from the extractmixture, or to remove the insolubles followed by a low molecularfraction therefrom, before the next anion exchange resin-adsorbing stepis carried out. For example, the extract mixture in which insolubes arecontained may be centrifuged to remove the insolubes, and the resultingsupernatant may be used in the next anion exchange resin-adsorbing step.Alternatively, the supernatant obtained by centrifuging the extractmixture containing insolubes may be dialyzed to remove a low molecularfraction (preferable a fraction of substances having a molecular weightof 3500 or less), and the resulting dialyzate may be used in the nextanion exchange resin-adsorbing step.

As an anion exchange resin which may be used in the anion exchangeresin-adsorbing step, a known anion exchange resin, for example,diethylaminoethyl (DEAE) cellulose or triethylaminoethyl (TEAE)cellulose, may be used.

An eluting solution used in the eluting step may be appropriatelyselected in accordance with an anion exchange resin used in the anionexchange resin-adsorbing step. For example, an aqueous solution ofsodium chloride may be used.

A fraction eluted by the eluting step may be used as the activeingredient of the immune enhancing agent of the present invention as itis, i.e., without purification. However, the fraction eluted by theeluting step usually contains salts derived from the eluting solution,and therefore, it is preferable to dialyze the fraction and remove thesalts.

The adsorption fraction of the extract mixture by an anion exchangeresin, as the active ingredient in the immune enhancing agent or theagent for promoting a recovery from stress according to the presentinvention, exhibits the following physicochemical properties.

-   (1) Carbohydrate content: The content of carbohydrates is 60% to 72%    (preferably 62% to 70%) in glucose equivalent by a phenol-sulfuric    acid method.-   (2) Protein content: The content of proteins is 28% to 40%    (preferably 30% to 38%) in albumin equivalent by a copper-Folin    method.-   (3) Carbohydrate composition: Glucose 61 μg/mg, mannose 3.3 μg/mg,    and galactose 2.0 μg/mg.-   (4) Amino acid composition: Aspartic acid and asparagine 10.35 mol    %, threonine 5.83 mol %, serine 6.27 mol %, glutamic acid and    glutamine 10.49 mol %, glycine 8.55 mol %, alanine 9.19 mol %,    valine 6.88 mol %, ½-cystine 0.60 mol %, methionine 1.49 mol %,    isoleucine 5.36 mol %, leucine 9.25 mol %, tyrosine 2.55 mol %,    phenylalanine 4.05 mol %, lysine 5.17 mol %, histidine 2.18 mol %,    arginine 4.44 mol %, tryptophan 1.82 mol %, and proline 5.54 mol %.-   (5) Isoelectric point: The isoelectric point of the main band is    around 5.85 by an isoelectric focusing method.-   (6) Nuclear magnetic resonance analysis (NMR)-   (i) ¹H one-dimensional NMR analysis: The spectrum is shown in    FIG. 7. As to the conditions for measurement, see Example 9(6)(i).-   (ii) ¹³C one-dimensional NMR analysis: The spectra are shown in    FIGS. 8 and 9. As to the conditions for measurement, see Example    9(6)(ii).-   (7) Circular dichroism analysis: The spectrum is shown in FIG. 10.    As to the conditions for measurement, see Example 9(7).-   (8) Optical rotation: Optical rotation measured at 25° C. is 42.-   (9) Infrared spectroscopic analysis: The spectrum is shown in    FIG. 11. As to the conditions for measurement, see Example 9(9).-   (10) Ultraviolet spectroscopic analysis (UV): The spectrum is shown    in FIG. 12. As to the conditions for measurement, see Example 9(10).-   (11) Electron spin resonance (ESR): The spectra are shown in FIGS.    13 and 14. As to the conditions for measurement, see Example 9(11).-   (12) Viscosity: The reduced viscosity at 30° C. is 108.-   (13) Molecular weight: The molecular weight of the major component    is 2000 kDa.-   (14) Elemental analysis: The contents of carbon (C), hydrogen (H),    nitrogen (N), sulfur (S), phosphorus (P), and chlorine (Cl) are    41.3%, 6.0%, 5.1%, 1.0%, 0.052%, and 0.16%, respectively.-   (15) Estimated content of α-glucan: 71% (with respect to all    carbohyderates).-   (16) Endotoxin content: 2.5 ng/mg

In the immune enhancing agent or the agent for promoting a recovery fromstress according to the present invention, the adsorption fraction ofthe extract mixture by an anion exchange resin, as the activeingredient, may be administered to an animal (preferably a mammal,particularly a human) with a pharmaceutically or veterinarily acceptableordinary carrier or diluent. The immune enhancing composition(preferably immune enhancing pharmaceutical composition) or thecomposition for promoting a recovery from stress (preferablypharmaceutical composition for promoting a recovery from stress)according to the present invention contains the adsorption fraction ofthe extract mixture by an anion exchange resin, as the activeingredient, and a pharmaceutically or veterinarily acceptable ordinarycarrier or diluent.

The active ingredient in the immune enhancing agent of the presentinvention, i.e., the adsorption fraction of the extract mixture by ananion exchange resin, exhibits an immuno-enhancing activity. Theimmuno-enhancing activity includes, for example, an antitumor activity[for example, an activity to extend a survival time in a subject withcancer, an anti-primary tumor activity (particularly an activity toinhibit a primary tumor proliferation), or an antimetastasis activity(particularly an activity to inhibit a metastatic focus proliferation)],an activity to produce an induction of a killer activity (particularlyan activity to produce an induction of a killer activity of anintestinal lymphocyte), an activity to enhance recognition of a tumorcell, an activity to enhance a gene expression of interleukin 12(IL-12), and an activity to increase a serum IAP value. The immuneenhancing agent of the present invention has an antitumor activity, suchas an anti-primary tumor activity (particularly an activity to inhibit aprimary tumor proliferation) or an antimetastasis activity (particularlyan activity to inhibit a metastatic focus proliferation), and thus itmay be used for treating or preventing cancer, such as a primary tumoror metastatic foci.

Therefore, the active ingredient in the present invention, i.e., theadsorption fraction of the extract mixture by an anion exchange resin,may be administered alone or, preferably, together with apharmaceutically or veterinarily acceptable ordinary carrier or diluentto a subject in need of an immune enhancement in an amount effectivetherefor.

Further, the active ingredient in the present invention, the adsorptionfraction of the extract mixture by an anion exchange resin, may be usedin the manufacture of an immune enhancing composition (preferably immuneenhancing pharmaceutical composition), an immune enhancing health food(preferably immune enhancing functional food), or an oral hygieniccomposition for an immune enhancement.

The active ingredient in the agent of the present invention forpromoting a recovery from stress, i.e., the adsorption fraction of theextract mixture by an anion exchange resin, exhibits an activity ofpromoting a recovery from stress.

Therefore, the active ingredient in the present invention, theadsorption fraction of the extract mixture by an anion exchange resin,may be administered alone or, preferably, together with apharmaceutically or veterinarily acceptable ordinary carrier or diluentto a subject in need of promoting a recovery from stress in an amounteffective therefor.

Further, the active ingredient in the present invention, the adsorptionfraction of the extract mixture by an anion exchange resin, may be usedin the manufacture of a composition for promoting a recovery from stress(preferably pharmaceutical composition for promoting a recovery fromstress), a health food for promoting a recovery from stress (preferablyfunctional food for promoting a recovery from stress), or an oralhygienic composition for promoting a recovery from stress.

Generally, when stress is loaded to an animal once or during a certainperiod, an immune activity in the animal is decreased but the immuneactivity is spontaneously recovered after a release of the stress. The“activity of promoting a recovery from stress” as used herein means anactivity which promotes the recovery of an immune activity during aperiod of an immune activity convalescence after a release of stress, incomparison with the spontaneous recovery.

The agent of the present invention for promoting a recovery from stresscan be administered at any time, so long as it can promote the recoveryof an immune activity briefly lowered by stress. It can be administered,for example, before a loading of stress, during a loading of stress,and/or during a period of an immune activity convalescence after arelease of stress.

In this connection, the “activity of promoting a recovery from stress”in the present invention is different from the above-described mere“immuno-enhancing activity” found by the present inventor. Generally,the “immuno-enhancing activity” means an activity in which anenhancement of an immune activity is observed by administering an activeingredient having such an activity in comparison with a state before theadministration, that is, an activity of enhancing an immune activity perse. The state before the administration may be a state in which animmune activity is natural or lowered by stress. In contrast, the“activity of promoting a recovery from stress” in the present inventionis an activity which promotes the recovery of an immune activity duringa period of an immune activity convalescence, as described above, thatis, an activity of enhancing the speed of recovery of an immuneactivity. When the agent of the present invention for promoting arecovery from stress is administered, the speed of recovery of an immuneactivity is increased in comparison with the case in which the agent ofthe present invention for promoting a recovery from stress is notadministered.

Further, in the “immuno-enhancing activity”, enhancement of an immuneactivity is directly observed when administering an active ingredienthaving such an activity. In contrast, when administering the activeingredient of the agent of the present invention for promoting arecovery from stress (i.e., the adsorption fraction of the extractmixture by an anion exchange resin) to a subject animal before a loadingof stress, the recovery of an immune activity is promoted during aperiod of an immune activity convalescence, even if the adsorptionfraction of the extract mixture by an anion exchange resin is notadministered during a loading of stress and during a period of an immuneactivity convalescence. With respect to the point, the “activity ofpromoting a recovery from stress” in the present invention is differentfrom the “immuno-enhancing activity” in the present invention.

The formulation of the immune enhancing agent or agent for promoting arecovery from stress of the present invention is not particularlylimited to, but may be, for example, oral medicines, such as powders,fine particles, granules, tablets, capsules, suspensions, emulsions,syrups, extracts or pills, or parenteral medicines, such as injections,liquids for external use, ointments, suppositories, creams for topicalapplication, or eye lotions.

The oral medicines may be prepared by an ordinary method using, forexample, fillers, binders, disintegrating agents, surfactants,lubricants, flowability-enhancers, diluting agents, preservatives,coloring agents, perfumes, tasting agents, stabilizers, humectants,antiseptics, antioxidants, such as sodium alginate, starch, corn starch,saccharose, lactose, glucose, mannitol, carboxylmethylcellulose,dextrin, polyvinyl pyrrolidone, crystalline cellulose, soybean lecithin,sucrose, fatty acid esters, talc, magnesium stearate, polyethyleneglycol, magnesium silicate, silicic anhydride, or synthetic aluminumsilicate.

The parenteral administration may be, for example, an injection such asa subcutaneous or intravenous injection, or a per rectum administration.Of the parenteral formulations, an injection is preferably used.

When the injections are prepared, for example, water-soluble solvents,such as physiological saline or Ringer's solution, water-insolublesolvents, such as plant oil or fatty acid ester, agents for renderingisotonic, such as glucose or sodium chloride, solubilizing agents,stabilizing agents, antiseptics, suspending agents, or emulsifyingagents may be optionally used, in addition to the active ingredient.

The immune enhancing agent or agent for promoting a recovery from stressof the present invention may be administered in the form of a sustainedrelease preparation using sustained release polymers. For example, theimmune enhancing agent of the present invention may be incorporated to apellet made of ethylenevinyl acetate polymers, and the pellet may besurgically implanted in a tissue to be treated.

The immune enhancing agent or agent for promoting a recovery from stressof the present invention may contain the adsorption fraction of theextract mixture by an anion exchange resin in an amount of, but is by nomeans limited to, 0.01 to 99% by weight, preferably 0.1 to 80% byweight.

A dose of the immune enhancing agent or agent for promoting a recoveryfrom stress of the present invention is not particularly limited, butmay be determined dependent upon the kind of disease, the age, sex, bodyweight, or symptoms of the subject, a method of administration, or thelike. The immune enhancing agent or agent for promoting a recovery fromstress of the present invention may be orally or parenterallyadministered.

The agent of the present invention may be administered as a medicamentor in various forms, for example, eatable or drinkable products such ashealth foods (preferably functional foods) or feeds. The term “foods” asused herein includes drinks.

It is known that food has (1) a function as nutrients (the firstfunction), (2) a function which appeals to five senses in a human (thesecond function), and (3) a function which affects a favorable influenceon health, physical ability, or mental condition in a human (the thirdfunction). The third function regulates various physiological systems,such as the digestive system, circulatory system, endocrine system,immune system, or nervous system, and affects a favorable influence onthe maintaining or recovery of health. The term “health food” as usedherein means food which provides, or is expected to provide, one or moreeffects on health. The term “functional food” as used herein meansprocessed or designed food such that the above various biologicalregulatory functions (i.e., functions which regulate physiologicalsystems such as the digestive system, circulatory system, endocrinesystem, immune system, or nervous system) may be fully expressed.

Further, the agent of the present invention may be administered in theform of an oral hygienic composition which is temporarily kept in themouth but then spat out, retaining almost none of the components, forexample, a dentifrice, a mouthwash agent, a chewing gum, or acollutorium, or in the form of an inhalation drawn in through the nose.More particularly, the adsorption fraction of the extract mixture by ananion exchange resin may be added to a desired food including a drink, afeed, a dentifrice, a mouthwash agent, a chewing gum, a collutorium, orthe like as an additive, such as a food additive.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples.

Example 1 Preparation of an Adsorption Fraction of an Extract Mixturefrom Mycelia of Tricholoma matsutake FERM BP-7304 by an Anion ExchangeResin

After a 7 t-culture tank containing 3.5 t of sterile medium (3% glucoseand 0.3% yeast extract, pH 6.0) was inoculated with mycelia ofTricholoma matsutake FERM BP-7304, cultivation was performed withshaking at 25° C. for 4 weeks. The resulting broth was filtered with afilter cloth to separate mycelia. The mycelia were washed with distilledwater.

After 30 L of purified water was added to a portion (about 1 kg) of theresulting mycelia, an extraction treatment was performed in a water bathat 98° C. for 3 hours while stirring. The whole was cooled andcentrifuged at 8000 rpm for 30 minutes to obtain a supernatant A₁. After30 L of purified water was added to the remaining pellets, extractionand centrifugation were performed in a similar manner as described aboveto obtain a supernatant A₂.

After 20 L of 0.5 mol/L sodium hydroxide solution was added to theremaining pellets, extraction was carried out at 25° C. for 1 hour, andthe whole was centrifuged to obtain a supernatant B₁. After 1.0 mol/Lsodium hydroxide solution was added to the remaining pellets, extractionand centrifugation were performed in a similar manner to obtain asupernatant B₂. The resulting supernatants B₁ and B₂ were combined andadjusted to pH 7.0 by adding 1.0 mol/L HCl (hereinafter referred to assupernatant B).

After the supernatants A₁, A₂, and B were combined, the resultingmixture (hereinafter referred to as extract mixture M) was put into adialysis tube (fractioning molecular weight=3500), and dialyzed inflowing water for 48 hours. The inner part of the dialyzate wascollected and dried by a lyophilizer to obtain approximately 70 g ofwhite dry powder.

A portion (10 g) of the resulting powder was dissolved in 500 mL of 50mmol/L tris-HCl buffer (pH 7.0), and the solution was applied to adiethylaminoethyl (DEAE) sephacel (Pharmacia) column which had beenequilibrated with the same buffer to obtain a fraction which passedthrough the column, as a non-adsorption fraction M1. After the columnwas washed with the tris-HCl buffer, a 50 mmol/L tris-HCl buffer (pH7.0) containing 0.5 mol/L of sodium chloride was applied to the columnto obtain an eluted fraction, as an adsorption fraction M2.

The resulting M21 fraction and M2 fraction were dialyzed in distilledwater for injection at 4° C. for 48 hours. The inner part of thedialyzate was lyophilized to obtain powder. The yields (with respect toa dry weight of mycelia) of the M1 and M2 fractions were 7% and 13%,respectively.

Example 2 Evaluation of the Adsorption Fraction M2 in Double GraftedTumor System

In the present example, an immuno-enhancing activity of the M2 fractionprepared in Example 1 was evaluated by a double grafted tumor system asan artificial model for a metastasis.

More particularly, 7-week-old male BALB/c mice (one group consisting of7 mice; Japan SLC) and syngeneic Meth-A fibrosarcomas were used. Eachmouse received simultaneous intradermal inoculations of Meth-A cells inthe right (2×10⁶) and left (4×10⁵) flanks. After three days from theinoculation [a tumor in the right flask (regarded as a primary focus)became large so that the tumor could be handled by the fingers], 5 mg ofthe M1 or M2 fraction prepared in Example 1 was repetitivelyadministered into the right tumor for 3 days, and the growth of the lefttumor (regarded as a metastatic focus) and the right tumor was observedfor 21 days. Instead of the M1 or M2 fraction, physiological saline wasadministered to a control group.

The results are shown in FIGS. 1 and 2 and Table 1.

FIG. 1 is a graph showing a time course for a tumor diameter(mean±standard deviation) of the right tumor. FIG. 2 is a graph showinga time course for a tumor diameter (mean±standard deviation) of the lefttumor. In the FIGS. 1 and 2, white circles represent the results in thecontrol group, black squares represent those for the M1 fraction, andwhite triangles represent those for the M2 fraction.

Table 1 shows each tumor diameter (mean±standard deviation; unit=mm) andtumor weight (mean±standard deviation; unit=g) of the right and lefttumors after 21 days from the inoculation.

Administration of the M2 fraction significantly inhibited the growth ofthe left and right tumors, in comparison with the control group. Theeffect was remarkable in the right tumor to which the M2 fraction wasdirectly injected. The growth of the left tumor without injection wasalso inhibited, and thus it was considered that the effect in the lefttumor was caused by a mechanism via immunity (cytokines). TABLE 1 Righttumor (2 × 10⁶ cells) Left tumor (4 × 10⁵ cells) Diameter WeightDiameter Weight (mm ± S.D.) (g ± S.D.) (mm ± S.D.) (g ± S.D.) Control23.5 ± 1.7 4.7 ± 1.0 20.3 ± 1.5 3.0 ± 1.0 M1 17.1 ± 1.5 1.8 ± 0.5 17.1 ±3.4 2.1 ± 1.1 M2  9.7 ± 3.2 0.6 ± 0.4 11.8 ± 2.5 0.7 ± 0.5

Example 3 Measurement of Serum IAP Level

In the present example, a change of serum IAP (immunosuppressive acidicprotein) levels by administration of the M2 fraction was observed. It isknown that the serum IAP is produced by activated macrophages, and thusthe serum IAP level may be used as an indicator of a macrophageactivation.

More particularly, 5 mg of the M1 or M2 fraction prepared in Example 1was intradermally injected to BALB/c mice, blood was collected withtime, and the serum IAP levels therein were measured by a SRID (singleradial immunodiffusion) method (J. Clausen, translated by Sai Sasaki andTakashi Murachi, “Immunological identification methods”, Tokyo KagakuDojin, 18-21, 1973).

The results are shown in FIG. 3. In FIG. 3, white circles represent theresults for the M1 fraction, and black circles represent the results forthe M2 fraction.

As shown in FIG. 3, 430 μg/mL of IAP was produced by administration ofthe M2 fraction.

Example 4 Evaluation for Activity of the Adsorption Fraction M2 forInhibiting Tumor (Sarcoma 180) Proliferation

As an animal subject, female ICR mice (CLEA Japan, Inc.) were used. As atumor, sarcoma 180 cells maintained in the peritonium of a female ICRmouse in Biomedical Research Laboratories, Kureha Chemical Industry Co.Ltd., were used. More particularly, sarcoma 180 cells (1×10⁶) weretransplanted at an axilla of 5-week-old female ICR mice (one groupconsisting of 10 mice). From the day after the transplanting, apredetermined amount (1.0 mg/kg, 10 mg/kg, or 50 mg/kg) of theadsorption fraction M2 obtained in Example 1 was intraperitoneallyadministered every other day, for 10 times in total. On the 25th dayafter the transplantation, mice were sacrificed, tumor nodes were taken,and the weights were measured. Physiological saline was administered tothe mice of the control group.

The ratio of proliferation inhibition (unit=%) was calculated by thefollowing equation:

-   -   [Ratio of proliferation inhibition (%)]={(Wc-W)/Wc}×100 wherein        W is an average weight (unit=g) of the node taken from the group        treated with the sample, and Wc is an average weight (unit=g) of        the node taken from the group treated with the physiological        saline.

The results are shown in Table 2. As apparent from Table 2,proliferation was significantly inhibited by administering the M2fraction. TABLE 2 Ratio of proliferation No. Sample/Dose inhibition (%)1 M2 fraction/1.0 mg/kg 71% 2 M2 fraction/10 mg/kg 81% 3 M2 fraction/50mg/kg 48%

Example 5 Evaluation of the Adsorption Fraction M2 for InfluenceProducing Induction of Killer Activity of Intestinal Lymphocytes

The M1 fraction, the M2 fraction, and the extract before fractionationthereof (i.e., lyophilized powder of the extract mixture M), prepared inExample 1, were orally administered to mice, and an influence producingan induction of a killer activity of intestinal lymphocytes wasexamined. The activity of cells from a mesenterium lymph node wasevaluated by a method of Harada et al. (Harada M. et al., Cancer Res.,55, 6146-6151, 1995).

More particularly, biological activities were examined by taking cellsof a mesenterium lymph node from a mouse to which tumor cells had beenimplanted at a cecal wall, and measuring a killer activity obtained whenthe tumor cells were re-stimulated in a test tube. The tumor cells usedin this Example were mouse leukemic cells P815 and B7/P815 which wereoriginally supplied from Dr. Mamoru Harada, Medical Institute ofBioregulation, Kyushu University (currently, Department of Medicine,Kurume University), and maintained in an RPMI 1640 medium containing 10%bovine fetal serum which had been heated at 56° C. for 30 minutes, atBiomedical Research Laboratories, Kureha Chemical Industry Co. Ltd.Female DBA/2 mice were purchased from Japan SLC and used in experimentsat 8 weeks-old after pre-breeding.

The mice were anesthetized by intraperitoneally administering 50 mg/kgof Nembutal and fixed. An abdomen was opened by scissors and tweezers, acecum taken out, B7/P815 cells (1×10⁶/50 μL) were implanted at a cecalwall, using 1 mL syringe equipped with a ⅛ G dental needle (Hata-jirushiMotoki Syringe Needle), and then the abdomen was closed by anatomicalstaplers. The mice which had recovered from anesthetization were putinto a breeding cage, and bred under ordinary breeding conditions. Fromthe day after the implantation of the tumor cells, each sample wasorally administered for 10 days in succession, using a probe for an oraladministration. A group of mice contained 10 mice.

On the day after the last administration day, the mice were sacrificed.Then, a lymph node of a mesenterium was aseptically taken out, put on asterilized dish containing a Hanks balanced salt solution, teased byscissors and tweezers, and passed through a mesh to prepare asingle-cell suspension of lymphocyte cells. The cells were washed withan RPMI 1640 medium containing a 10% bovine fetal serum which had beenheated at 56° C. for 30 minutes three times. Then, a concentration ofcells was adjusted to 5×10⁶/mL with an RPMI 1640 medium containing a 10%bovine fetal serum which had been heated at 56° C. for 30 minutes,5×10⁻⁵ mol/L of 2-mercaptoethanol, 20 mmol/L of4-(2-hydroxyethyl)-1-piperazine ethanesulfonate, and 30 μg/mL ofgentamicin and used as effector cells.

Stimulating cells were prepared as follows: P815 cells or B7/P815 cellswere suspended in an RPMI 1640 medium, so that a concentration became5×10⁶/mL, and mitomycin C (Sigma) was added thereto so that aconcentration thereof was 50 μg/mL. After a reaction was performed in a5% carbon dioxide gas incubator for 30 minutes, the cells were washedwith an RPMI 1640 medium containing a 10% bovine fetal serum which hadbeen heated at 56° C. for 30 minutes three times, and a concentration ofcells was adjusted to 1×10⁵/mL.

A mixed lymphocyte tumor cell reaction (MLR) was examined under thefollowing conditions.

The effector cells (0.1 mL) and/or the stimulating cells (0.1 mL) wereput on a 96-well culturing flat-bottomed microplate (Falcon 3072; BectonDickinson Labware, USA), cultured in a 5% carbon dioxide gas incubatorat 37° C. for 3 days, and recovered on a filter. When both the effectorcells and the stimulating cells were put into the microplate, a ratio ofthe cells (the number of the effector cells/the number of thestimulating cells) was 12.5. In this examination system, the effectorcells functioned as lymphocytes in the mixed lymphocyte tumor cellreaction, and the stimulating cells functioned as tumor cells in themixed lymphocyte tumor cell reaction. Before 8 hours of completing theculturing, 37 kBq of ³H-thymidine (Amersham Japan) was added to eachwell of the plate. The harvested cells were thoroughly washed with 5%trichloroacetic acid, dried, and put into a vial for liquid. Afteradding a liquid scintillator, the radioactivity was measured by a liquidscintillation counter.

A stimulation index (S.I.) was calculated by an equation:[S.I.]=(Bmix-Bs)/(Be-Bs)wherein Bmix is a radioactivity (unit=Bq) of a group of the mixedculture of the effector cells and the stimulating cells, Bs is aradioactivity (unit=Bq) of a group of the single culture of thestimulating cells, and Be is a radioactivity (unit=Bq) of a group of thesingle culture of the effector cells.

An MLR-cell-mediated Cytotoxicity (MLR-CMC) was examined under thefollowing conditions.

The effector cells (1.0 mL) and the stimulating cells (1.0 mL) were puton a 24-well culturing microplate (Culture Clastar; Costar 3524; CorningInc., USA) at a ratio of the cells (the number of the effector cells/thenumber of the stimulating cells) of 12.5, and cultured in a 5% carbondioxide gas incubator for 3 days. After the culturing was completed, thecells were recovered and washed three times with an RPMI 1640 mediumcontaining 10% bovine fetal serum which had been heated at 56° C. for 30minutes, and the number of only the effector cells in the cellsuspension was counted, using a microscope, so that a concentration ofthe effector cells was adjusted to 2.5×10⁶/mL.

P815 cells were reacted with sodium chromate (Amersham Japan) at 37° C.for 20 minutes. Unreacted radioactive substances were removed by washingwith an RPMI 1640 medium containing 10% bovine fetal serum which hadbeen heated three times at 56° C. for 30 minutes, and a concentration oftumor cells labeled with radioactive chromium was adjusted to 5×10⁴/mL.

0.1 mL of the effector cells or a double-diluted series thereof and 0.1mL of tumor cells labeled with radioactive chromium were put into a testtube, and reacted in a 5% carbon dioxide gas incubator at 37° C. for 4hours. After the reaction was completed, 1.5 mL of an RPMI 1640 mediumcontaining a 10% bovine fetal serum, which had been heated at 56° C. for30 minutes, was added to each test tube, and thoroughly mixed by amixer. The whole was centrifuged at 12,000 rpm for 5 minutes at 4° C. toobtain a supernatant, and a radioactivity was measured by a gammacounter.

A specific lysis (S.L.; unit=%) was calculated by an equation:[S.L.(%)]={(B-Bf)/(Bmax-Bf)}×100wherein B is a radioactivity (unit=Bq) of a supernatant of anexperimental group, Bf is a radioactivity (unit=Bq) of a supernatant ofa spontaneously releasing group, and Bmax is a radioactivity (unit=Bq)of a supernatant of a maximum releasing group. The spontaneouslyreleasing group means a group of culturing only the tumor cells labeledwith radioactive chromium, and the maximum releasing group means a groupof culturing tumor cells labeled with radioactive chromium treated with5% Triton X100.

The results of the influence on the MLR-CMC for the samples prepared inExample 1 are shown in Table 3, when the ratio of the number of theeffector cells/the number of the stimulating cells was 12.5. In Table 3,“*” means that there was a significant difference of p<0.01 on the basisof the control group. To control groups, 0.2 mL of purified water wasorally administered. As apparent from Table 3, the M2 fraction had theactivity for inducing the MLR-CMC, and the activity exhibited adose-dependency. TABLE 3 Experimental MLR-CMC (% to the No. Sample/Dosecontrol group) 1 Extract (before fractionation)/ 153* 250 mg/kg M1/250mg/kg 109  M2/250 mg/kg 181* 2 M2/63 mg/kg 131* M2/125 mg/kg 159* M2/250mg/kg 197*

Example 6 Evaluation of the Adsorption Fraction M2 for Activity toEnhance Gene Expression of IL-12

The gene expression of interleukin 12 (IL-12) in cells from amesenterium lymph node was measured by a reversetranscriptase-polymerase chain reaction (RT-PCR) method in accordancewith a method of Harada et al. (Harada M. et al., Cancer Res., 58,3073-3077, 1998).

More particularly, tumor cells were implanted in mice at a cecal wallthereof in a manner similar to that in Example 5. Instead of tumorcells, physiological saline was injected to a cecal wall to prepare shammice. To the tumor-implanted mice, the sham mice, and normal mice, 250mg/kg/day of the M2 fraction or distilled water was administered. After11 days from the implantation, mice were sacrificed, and thenmesenterium lymph nodes were taken, immediately frozen in liquidnitrogen, and kept at −80° C. until analysis.

The following procedures were carried out in accordance with protocolsattached to commercially available kits or reagents, unless otherwisespecified. Total RNA was prepared from each sample by a reagent for RNApreparation (TRIzol reagent; Life Technologies, Inc., USA). Theresulting total RNAs were reverse-transcribed using a reversetranscriptase (Superscript reverse transcriptase; Life Technologies,Inc.), random hexamers, and deoxyribonucleoside triphosphates, tosynthesize cDNAs. The cDNAs were amplified in the presence of Taq DNApolymerase (Perkin-Elmer Co., USA), deoxyribonucleoside triphosphates,and a set of primers, using a PCR thermal cycler (Perkin-Elmer PCRThermal Cycler TP-2000; Perkin-Elmer Co.).

In the above PCR, cycle numbers in the case of IL-12 p40 and β-actinwere 30 and 36, respectively. As to the primer set, oligonucleotidesconsisting of the base sequences of SEQ ID NOS: 1 and 2 were used assense and antisense primers for IL-12 p40. Oligonucleotides consistingof the base sequences of SEQ ID NOS: 3 and 4 were used as sense andantisense primers for β-actin.

The PCR products were electrophoresed using an agarose gel containingethidium bromide, and digitized using a multi-analyst system (Bio-RadMulti-Analyst System).

The results are shown in FIG. 4. In FIG. 4, graph (A) shows the resultsin normal mice, graph (B) shows the results in sham mice, and graph (C)shows the results in tumor-implanted mice. In FIG. 4, a rate of theIL-12 p40 gene expression is represented by a percentage with respect tothe normal and distilled water-administered mice group.

The IL-12 gene expression was enhanced in the tumor-implanted anddistilled water-administered mice group, in comparison with the normaland distilled water-administered mice group. The M2 fraction littleaffected the IL-12 gene expression in the normal mice, but significantlypromoted it in the tumor-implanted mice. Further, the M2 fractionpromoted it slightly in the sham mice.

Example 7 Evaluation for Extension of Survival Time and Effect ofAnit-IL-12 Antibody Treatment in Mice in Which Tumor was Immunized andthe M2 Adsorption Fraction was Orally Administered

In the present example, an influence of the M2 fraction on an antitumoractivity was examined. When B7/P815 cells were implanted in a mousececum, a tumor proliferated at first, but tumor regression occurredafter 14 days from the implantation, and all mice survived. By contrast,when P815 cells were implanted at a mouse cecal wall, tumor cellsproliferated and metastasized, and all mice died of tumor. Therefore,B7/P815 tumor cells (5×10⁵ cells/dose) were immunized in a cecum ofDBA/2 mice three times at intervals of two weeks, and after two weeksfrom the last injection, P815 cells (1×10⁵ cells) were implanted in acecum thereof, and the survival time of each mouse was observed. In thisconnection, with respect to the proliferation of P815 tumor cells in acecum, the group in which B7/P815 cells were immunized in a cecum wassuperior to the subcutaneously-immunized group.

The results are shown in Table 4 and FIG. 5. In Table 4 and FIG. 5, thegroup I is a nonimmunized and distilled water-administered group; thegroup II is an immunized and distilled water-administered group; thegroup III is an immunized and M2-administered group in which the M2fraction (250 mg/kg) was orally administered every day during theimmunized period; and the group IV is an immunized, M2-administered, andanti-IL-12 antibody-treated group in which the M2 fraction (250 mg/kg)was orally administered every day during the immunized period and ananti-IL-12 antibody (200 μg/dose) was intravenously injected before 12days, 7 days, and 2 days from the implantation of P815 cells. In Table4, “SD” means a standard deviation, “T” in “T/C” means a survival timein the test groups (i.e., groups II to IV), “C” means a survival time inthe control group (i.e., group I), and “*” means that there was asignificant difference of p<0.01 with respect to the group II. TABLE 4Survival time Group mean ± SD (T/C) × 100 I 13.2 ± 0.8 — II 16.3 ± 1.2100 III 20.8 ± 1.9  128* IV 17.7 ± 1.2 109

As shown in FIG. 5, the survival time was extended by approximately 120%in the immunized and distilled water-administered group (group II), incomparison with the nonimmunized and distilled water-administered group(group I). The survival time was significantly extended by 128% in theimmunized and M2-administered group (group III), in comparison with theimmunized and distilled water-administered group (group II). In theimmunized, M2-administered, and anti-IL-12 antibody-treated group (groupIV), the survival time was attenuated to 109% of that in the immunizedand distilled water-administered group (group II).

In a similar fashion, colon26 tumor cells treated with mitomycin C wereimmunized to BALB/c mice at a cecal wall three times at intervals of twoweeks, and colon26 living cells were implanted. The results are shown inTable 5 and FIG. 6. The groups I to IV in Table 5 and FIG. 6 have thesame meanings in Table 4 and FIG. 5. TABLE 5 Survival time Group mean ±SD (T/C) × 100 I 16.8 ± 1.5 — II 19.4 ± 1.6 100 III 23.3 ± 1.3  120* IV20.4 ± 1.4 105

In the nonimmunized and distilled water-administered group (group I),almost all of the mice died of cachexia, intestinal obstruction causedby tumor proliferation, or peritonitis. However, oral administration ofthe M2 fraction significantly extended the survival time of the mice.The effect was attenuated by the treatment of anti-IL-12 antibody. Theadministration of the M2 fraction to the nonimmunized mice did notaffect the survival time.

Example 8 Evaluation of the Adsorption Fraction M2 for Activity ofPromoting a Recovery from Stress

In the present example, the M2 fraction prepared in Example 1 was usedas a sample for evaluation. Further, after the sample for evaluation wasorally administered to mice for 10 days, restraint stress was loaded for18 hours, and then a natural killer (NK) cell activity was measuredafter a release of the stress to examine the effects of the sample.

More particularly, an aqueous solution of the sample for evaluation wasorally administered to 8-week-old male C57BL/6 mice (purchased fromCharles River Japan; 5 to 10 mice per a group) at a dose of 300mg/kg/day for 10 days in normal breeding cages. After the administrationfor 10 days, mice were transferred from the normal breeding cages to 50mL-capped polypropylene centrifuge tubes (catalog No. 2341-050; AsahiTechno Glass Corporation) with air vents so that a mouse was confined ina tube. The confined mice could not move in the tubes. The tubes inwhich mice were confined were placed in the cages and allowed to standfor 18 hours to load the mice with restraint stress. After the stressloading for 18 hours, mice were transferred from the tubes to thebreeding cages, and bred under ordinary breeding conditions.

After 7 days had passed from the release of the restraint stress, micewere sacrificed, and a natural killer (NK) cell activity was evaluatedby measuring a cytotoxic activity of lymphocytes against an NK-sensitivetumor cell strain YAC-1 in vitro, in accordance with a method ofGreenberg et al. (Greenberg A. H. et al., J.Exp.Psychol., 12, 25-31,1986) as follows.

Spleens and mesenterium lymph nodes were aseptically taken from mice andtransferred to a sterile petri dish containing a Hanks balanced saltsolution. The lymph nodes were teased with scissors and tweezers, andpassed through a mesh to prepare a suspension containing singlelymphocyte cells. The cells were washed three times with an RPMI 1640medium containing a 10% bovine fetal serum which had been heated at 56°C. for 30 minutes. Then, a concentration of cells was adjusted to5×10⁶/mL with an RPMI 1640 medium containing a 10% bovine fetal serumwhich had been heated at 56° C. for 30 minutes, 20 mmol/L of4-(2-hydroxyethyl)-1-piperazine ethanesulfonate, and 30 μg/mL ofgentamicin. The resulting cell suspension was used as an effector cell.

The YAC-1 cell used as a target cell was maintained in an RPMI 1640medium containing a 10% bovine fetal serum which had been heated at 56°C. for 30 minutes, at Biomedical Research Laboratories, Kureha ChemicalIndustry Co. Ltd. The YAC-1 cells were reacted with radioactive sodiumchromate (Amersham Japan) at 37° C. for 20 minutes. Unreactedradioactive sodium chromate was removed by washing three times with anRPMI 1640 medium containing a 10% bovine fetal serum which had beenheated at 56° C. for 30 minutes, and a concentration of tumor cellslabeled with radioactive chromium was adjusted to 5×10⁴/mL.

0.1 mL of the effector cell suspension or a double-diluted seriesthereof and 0.1 mL of the suspension of tumor cells labeled withradioactive chromium were put into a test tube, and reacted in a 5%carbon dioxide gas incubator at 37° C. for 4 hours. In this connection,to calculate a “specific lysis” described below, a suspension preparedby putting the tumor cells labeled with radioactive chromium and amedium into a test tube, and a suspension prepared by putting the tumorcells labeled with radioactive chromium and a detergent (Triton; a finalconcentration=0.05%) into a test tube were also reacted in a 5% carbondioxide gas incubator at 37° C. for 4 hours. After the reaction wascompleted, 1.5 mL of an RPMI 1640 medium containing a 10% bovine fetalserum, which had been heated at 56° C. for 30 minutes, was added to eachtest tube, and thoroughly mixed by a mixer. The whole was centrifuged at12,000 rpm for 5 minutes at 4° C. to obtain a supernatant, and theradioactivity was measured by a gamma counter.

A specific lysis (S.L.) was calculated by an equation:[S.L.]={(B-Bf)/(B _(max)-Bf)}×100wherein S.L. is a specific lysis (unit=%), B is a radioactivity(unit=Bq) of a supernatant of an experimental group, Bf is aradioactivity (unit=Bq) of a supernatant of a spontaneously releasinggroup, and B_(max) is a radioactivity (unit=Bq) of a supernatant of amaximum releasing group. The spontaneously releasing group means a groupof culturing only the tumor cells labeled with radioactive chromium, andthe maximum releasing group means a group of culturing tumor cellslabeled with radioactive chromium treated with Triton. The NK cellactivity was represented by “Lytic Units 30% (LU30)”, that is, a numberof cells which kill 30% tumor cells per 10⁷ cells of effector cells.

The results are shown in Table 6. As a control (a normal group), theabove procedures were repeated, except that distilled water was orallyadministered for 10 days, instead of the aqueous solution of the samplefor evaluation, and that the restraint stress for 18 hours was notloaded. Further, as a test for comparison, the above procedures wererepeated, except that distilled water was orally administered for 10days, instead of the aqueous solution of the sample for evaluation. Inthe column “Restraint” of Table 6, the symbols “(−)” and “(+)” mean“without restraint stress” and “with restraint stress”, respectively.

According to a significant test, the comparative group showed P<0.01with respect to the control group, and the M2 fraction-administeredgroup showed P<0.05 with respect to the comparative group. TABLE 6 GroupRestraint NK activity (LU30) Control (−) 46.6 ± 4.2 Comparative (+) 28.7± 2.4 M2 fraction-adminstered (+) 34.8 ± 4.4

Example 9 Examination of Physicochemical Properties of the AdsorptionFraction M2

Physicochemical properties of the M2 fraction prepared in Example 1 andthe m2 fraction, prepared in Referential Example 1 as described below,from fruit bodies of commercially available Tricholoma matsutake wereexamined. Measuring methods and the results will be described below.

(1) Determination of Carbohydrates

Carbohydrate content in the M2 fraction was determined by colorimetryusing a phenol-sulfuric acid method. The content of carbohydrates in theM2 fraction was 62% in glucose equivalent.

The above procedure described in Example 1 was repeated twice to obtaintwo kinds of M2 fraction. The contents of carbohydrates therein were 69%and 70%, respectively, determined by a similar method.

The content of carbohydrates in the m2 fraction was 35% in glucoseequivalent.

According to an iodostarch reaction, the M2 fraction and m2 fractionwere negative. The results suggested that carbohydrates other thanstarch were contained.

(2) Determination of Proteins

Protein content in the M2 fraction was determined by colorimetry using acopper-Folin method. The content of proteins therein was 38% in albuminequivalent.

The above procedure described in Example 1 was repeated twice to obtaintwo kinds of M2 fraction. The contents of proteins therein were 31% and30%, respectively, determined by a similar method.

The content of proteins in the m2 fraction was 65% in albuminequivalent.

(3) Analysis of the Carbohydrate Composition

Into a tube, 1.0 mg of the M2 fraction and 0.2 mL of 2 mol/Ltrifluoroacetic acid were charged, and hydrolyzed at 100° C. for 6hours. The reaction mixture was dried under a reduced pressure by anevaporator to obtain a residue. The residue was dissolved in 500 μL ofpurified water, and further diluted to a double volume or a ten-foldvolume with purified water. To 50 μL of this solution, 500 ng of heptosewas added as an internal standard substance, and the solution wasapplied to a high performance liquid chromatograph LC-9A (Shimadzu)equipped with a column TSK-gel Sugar AXGLC-9A (15 cm×4.6 mm ID; Tosoh)and a spectrophotometer RF-535 (Shimadzu) as a detector. The columntemperature was 70° C. The mobile phase was a 0.5 M potassium boratebuffer (pH 8.7), and the flow rate thereof was 0.4 mL/min. For theconditions of post-column labeling, 1% arginine/3% boric acid was usedas a reaction reagent, the flow rate was 0.5 mL/min., the reactiontemperature was 150° C., and the wavelengths for detection were EX 320nm and EM 430 nm.

The carbohydrate composition in the M2 fraction was as follows:

-   -   Glucose 61 μg/mg, mannose 3.3 μg/mg, and galactose 2.0 μg/mg, in        the order of descending content.

The carbohydrate composition in the m2 fraction was as follows:

-   -   Glucose 12.9 μg/mg, galactose 12.6 μg/mg, mannose 5.6 μg/mg,        fucose 3.5 μg/mg, and xylose 0.4 μg/mg, in the order of        descending content.        (4) Analysis of the Amino Acid Composition

Acidic hydrolysis was carried out as follows. Into a tube, 0.33 mg ofthe M2 fraction and 0.2 mL of 6 mol/L hydrochloric acid were charged,and hydrolyzed at 110° C. for 22 hours. The reaction mixture was driedunder a reduced pressure by an evaporator to obtain a residue. Theresidue was dissolved in 0.5 mL of purified water, and 50 μL of thesolution was used for an amino acid analysis.

Alkaline hydrolysis for analyzing tryptophan was carried out as follows.After 0.48 mg of the M2 fraction was added to a plastic tube, 100 μL of1% n-octyl alcohol-4.2 mol/L sodium hydrate solution containing 5 mg ofstarch soluble were further added thereto. After the plastic tube wasplaced into a glass tube, hydrolysis was carried out in the sealed andvacuum tube at 110° C. for 16 hours. The glass tube was cooled in airand opened. After the plastic tube was cooled in water, 1.0 mol/Lhydrochloric acid was added thereto to neutralize the mixture. Into thetube, 840 μL of purified water was added to adjust the total volume to1000 μL, and 50 μL thereof was used for an amino acid analysis.

The quantitative determination was performed by a ninhydrin colorimetryusing an amino acid analyzer L-8500 (Hitachi) as equipment.

The amino acid composition was as follows:

-   -   Aspartic acid and asparagine 10.35 mol %, threonine 5.83 mol %,        serine 6.27 mol %, glutamic acid and glutamine 10.49 mol %,        glycine 8.55 mol %, alanine 9.19 mol %, valine 6.88 mol %,        ½-cystine 0.60 mol %, methionine 1.49 mol %, isoleucine 5.36 mol        %, leucine 9.25 mol %, tyrosine 2.55 mol %, phenylalanine 4.05        mol %, lysine 5.17 mol %, histidine 2.18 mol %, arginine 4.44        mol %, tryptophan 1.82 mol %, and proline 5.54 mol %.        (5) Analysis of the Isoelectric Point

The M2 fraction was adjusted to 1 mg/mL. To (a) a mixture of 10 μL ofthe M2 fraction solution and 10 μL of purified water or (b) 20 μL of theM2 fraction solution (about 1.14 μg as an amount of proteins),saccharose was added so that the concentration thereof becameapproximately 40% (volume/volume), and then an electrophoresis wasperformed. The conditions of the electrophoresis were as follows:

-   Gel: IEF-PAGEmini (4%, pH 3-10; Tefco).-   Buffer for electrophoresis: (cathode) 0.04 mol/L sodium hydroxide    solution, (anode) 0.01 mol/L phosphate solution.-   Conditions for electrophoresis: The electrophoresis was performed at    100 V for 30 minutes, at 300 V for 20 minutes, and at 500 V for 40    minutes.-   PI marker: Each band was 1.35 g (Pharmacia).-   Staining: Silver staining.

A main band was around 5.85.

(6) Nuclear Magnetic Resonance Analysis (NMR)

The conditions for measurements were as follows.

(i) ¹H One-Dimensional NMR Measurement

After 800 μL of D₂O was added to 7 mg of the M2 fraction,ultrasonication was carried out for about 5 minutes in an attempt todissolve it. The whole was centrifuged, and the supernatant was used formeasurement. The conditions of the measurement were as follows.

As a detector, UNITY INOVA 600 (Varian) was used. An observationfrequency was 599.6 MHz (¹H nucleus). As a solvent, D₂O solution(concentration: saturated solution) was used. TSP 0.00 ppm (¹H) was usedas a standard. The temperature was 25° C. The repetition time was 7.0seconds (¹H nucleus). The number of accumulation was 256.

The resulting spectrum is shown in FIG. 7. Strong signals derived fromcarbohydrates were observed between 3.0-5.6 ppm. It was presumed thatthe M2 fraction contained many carbohydrates, because the signalintensities from carbohydrates were much stronger than those from aminoacids. In this connection, it is known that the signals observed between0.5-3.0 ppm are derived from side chains of amino acids. Further, NMRsignals from aromatic amino acids were observed between 6.6-7.6 ppm.

The estimated content of α glucan was 71%.

(ii) ¹³C One-Dimensional NMR Measurement

The M2 fraction was dissolved in D₂O/CD₃OD (725/25), so that aconcentration became approximately 20.5 mg/0.75 mL. The conditions ofthe measurement were as follows.

The measurement was performed at the frequency of 125.8 MHz. Thestandard was a deuterated methanol (δ=49 ppm). The temperature was 45°C. The observation width was 31.4 KHz. The data point was 64 K. Thepulse width was about 41°. The repetition time of pulse was 2.5 seconds.The number of accumulation was 4000. Measurement of decoupling wasperformed under conditions of ¹H complete decoupling.

The results are shown in FIGS. 8 and 9. Signals from carbohydrates andsignals from amino acids were observed. The signal intensities fromcarbohydrates were stronger than those from amino acids. Because themajor component of carbohydrates contained in the M2 fraction isglucose, it was considered that signals around 95-110 ppm were derivedfrom a carbon at the 1-position of glucose, signals around 105 ppm werefrom a carbon at the β1 position, and signals around 102 ppm and around99 ppm were from a carbon at the α1 position. From the results, at leastthree or more binding types were presumed. Signals around 63 ppm werederived from the 6-position. The fact that three signals were observedaround 63 ppm supports the above presumption, i.e., the M2 fraction hasthree or more binding types. Further, it is considered from signalsaround 70-80 ppm that the 4-position is involved in binding, and it ispresumed that an α1-4 bond and a β1-4 bond are contained in the M2fraction.

(7) Circular Dichroism Analysis (CD)

Water was added to approximately 3 mg of the M2 fraction, so that aconcentration became 2 mg/mL. Because a precipitate was observed, thewhole was centrifuged, and the supernatant was used for measurement. Theconditions for measurement were as follows.

As a detector, JASCOJ-500A was used. As a solvent, water was used. Theconcentration of proteins was approximately 2 mg/mL. The wavelength areawas 200 to 250 nm. The cell length was 1 mm. The temperature was roomtemperature (approximately 23° C.). The number of accumulation was 8.The measurement was performed under the above conditions.

The resulting CD spectrum is shown in FIG. 10. The CD value (verticalaxis) is ellipse angle (mdeg) . It was presumed that secondarystructures such as α-helix were a minor structure, and that unorderedstructure was a major structure.

(8) Optical Rotation

Optical rotation measured at 25° C was 42.

(9) Infrared Spectroscopic Analysis

Infrared spectroscopic analysis was carried out by a KBr method. Moreparticularly, 0.5 mg of the M2 fraction was uniformly mixed with 15 mgof KBr powder, and disks were formed by pressing and measured.

The resulting spectrum is shown in FIG. 11. This spectrum suggested thatpolysaccharides were contained in the M2 fraction.

(10) Ultraviolet Spectroscopic Analysis (UV)

The M2 fraction was dissolved in purified water (concentration=0.5 mg/10mL) and a UV was measured. As a detector, 2500PC (Shimadzu) was used.

The resulting ultraviolet and visible absorption spectrum is shown inFIG. 12. A weak absorption maximum was detected at 260 to 270 nm.

(11) Electron Spin Resonance (ESR)

The ESR of the sample was measured under a nitrogen atmosphere usingESP350E (Brucker). The conditions of the measurement are shown in Table7.

The results are shown in Table 8 and FIGS. 13 and 14. In FIGS. 13 and14, the phrase “Intensity (arb. units)” in the vertical axis means thatthe unit of “intensity” shown at the vertical axis is arbitrary. Asignal, which was considered to be derived from carbon radicals, wasobserved around g=2.004. Further, it was considered that signals aroundg=4.25 (Fe³⁺) and around g=2.03-2.05 were derived from transition metalions. TABLE 7 Conditions Broad Around g = 2 Measuring temperature RoomRoom temperature temperature Magnetic field 0˜1 T 339.0-359.0 mT sweeparea Modulation 100 kZ, 100 kZ, 0.5 mT 0.2 mT Microwave 10 mW, 0.2 mW,9.79 GHz 9.79 GHz Sweep time 167.772 s × 83.886 s × 1 time 10 times Timeconstant 163.84 ms 163.84 ms Number of data 4096 points 2048 pointspoints Cavity TM₁₁₀, cylinder TM₁₁₀, cylinder

TABLE 8 Index M2 fraction m2 fraction g value 2.0042 2.0039 Line width(mT) 0.68 0.67 Spin density (spins/g) 4.7 × 10¹⁶ 2.8 × 10¹⁶(12) Viscosity

After 0.5 g of the sample (the M2 fraction or m2 fraction) was dissolvedin 100 mL of purified water, and the whole was centrifuged at 10000 rpm.The supernatant was adjusted to 1.67 mg/mL by adding purified water,and- a reduced viscosity was measured at 30° C. by an Ostwaldviscometer. The reduced viscosities of the M2 and m2 fractions were 108η and 924 η, respectively.

(13) Molecular Weight

After the sample (the M2 fraction or m2 fraction) was dissolved inpurified water, so that the concentration was 2-3 mg/mL, gel filtrationwas carried out.

In the gel filtration, a feed pump LC-7A (Shimadzu) was used asequipment, an ultraviolet spectrophotometer detector SPD-6A (Shimadzu)was used as a detector, and TSKgel G3000SW (7.5 mm I.D.×30 cm; Tosoh)was used as a column. Further, the column temperature was roomtemperature, the mobile phase was 50 mmol/L phosphate buffer (pH 7.0)containing 0.15 mol/L sodium sulfate, the flow rate thereof was 0.8mL/min, and the wavelength for detection was 214 nm. A molecular weightwas calculated by extrapolating an elution time to a standard curveprepared from compounds with a known molecular weight.

In the M2 fraction, the molecular weight of the major component was 2000kDa, and other components of 4.0 kDa and 1.2 kDa were confirmed. In them2 fraction, the molecular weight of the major component was 2000 kDa,and other components of 7.0 kDa and 1.0 kDa were confirmed.

(14) Elemental Analysis

Carbon (C), hydrogen (H), and nitrogen (N) were measured using CHN coderTM-S (Yanako).

As to sulfur (S), phosphorus (P), and chlorine (Cl), after the samplewas combusted and decomposed, SO²⁻, PO₄ ³⁻, and Cl⁻ in an absorbingsolution were measured by an ion chromatography (IC) method, and theresulting values were converted to those of the elements. Moreparticularly, 1 mL of acetone was added to 0.1 g of the sample. After anintroduction of oxygen (3 Mpa), the whole was subjected to combustionand cooled in water for 30 minutes. After an absorbing solution (0.1mol/L NaOH) and a washing solution were combined and adjusted to 100 mL,the measurement was carried out using Dionex DX-300 type IC.

The results are shown in Table 9. TABLE 9 Content (%) Elements M2fraction m2 fraction carbon 41.3 40.4 hydrogen 6.0 6.0 nitrogen 5.1 8.0sulfur 1.0 0.22 phosphorus 0.052 0.096 chlorine 0.16 0.13(15) Estimated Content of α-Glucan

After the sample (the M2 fraction or m2 fraction) was dissolved in 0.5mol/L of an acetate buffer (pH 4.3), an amyloglucosidase solution (SigmaChem. Co., USA) was added to the solution. After the whole was shaken at60° C. for 30 minutes and adjusted to pH 4.5, glucoamylase was added.The whole was further shaken at 60° C. for 30 minutes. After thereaction, an amount of glucose in the resulting solution was measured bya glucose analyzer. The value obtained by subtracting the measured valuefrom an amount of glucose in a blank solution was regarded as an“estimated amount of α-glucan”.

In addition, after 1.0 mol/L of sulfuric acid was added to the sample,the whole was hydrolyzed at 100° C. for 18 hours, and then neutralized.An amount of glucose in the resulting solution was measured by a glucoseanalyzer, and the value was regarded as a “total amount of glucan”. Theestimated content of α-glucan was calculated as a percentage of the“estimated amount of α-glucan” with respect to the “total amount ofglucan”.

The estimated content of α-glucan in the M2 fraction was 71% withrespect to all carbohydrates, and that in the m2 fraction was 32% withrespect to all carbohydrates.

(16) Determination of Endotoxin

An amount of endotoxin was determined by a LAL (Limulus AmoebocyteLysate) reaction (Ohbayashi T. et al., Clin.Chim.Acta, 149, 55-65, 1985)using a commercially available measuring kit (Endospecy; SeikagakuCorp.), equipment (endotoxin-free), and reagents (Seikagaku Corp.).

More particularly, after the M2 fraction was dissolved in distilledwater, so that the concentration became an appropriate concentration, 50μL of the aliquot was added to a well in a 96-well microplate(endotoxin-free). The same volumes of a diluted series of an endotoxinstandard solution or distilled water were added to other wells of themicroplate. Then, 50 μL of an LAL solution (a reagent from Limulus) wasadded to each well thereof. After incubation at 37° C. for 30 minutes, adiazo coupling reagent was added. After color development, absorbance at545 nm (control=630 nm) was measured. An amount of endotoxin in the M2fraction calculated from a calibration curve of the standard solutionwas 2.5 ng/mg.

Example 10 Presumption of Active Structure of the M2 Fraction

In the present example, to presume an active structure of the M2fraction, the M2 fraction was chemically or enzymatically treated, theresulting preparation was orally administered to mice to which tumorcells had been implanted at a cecal wall, and the activity of cells froma mesenterium lymph node was evaluated by MLR and MLR-CMC.

More particularly, a peptide portion of the M2 fraction was preparedaccording to a method of Takasaki et al. (Takasaki S. et al., Methods inEnzymology, vol.83, Academic Press, New York, 263-268, 1982) to preparea fraction with a reduced content of proteins as follows. After the M2fraction (carbohydrates:proteins=68:32) was dissolved in hydrazineanhydride, the solution was added to a glass tube, and heated at 100° Cfor 24 hours in the sealed tube. The resulting solution was placed in adesiccator, and allowed to stand under reduced pressure to removehydrazine. After the dried product was dissolved in purified water, thesolution was applied to gel filtration using Sephadex G-25 (Pharmacia)to collect a high molecular fraction. The resulting fraction was appliedto an ion-exchange chromatography using diethylaminoethyl sephacel toobtain a fraction which passed through the column(carbohydrates:proteins=99:1).

To prepare a fraction with a reduced content of α-glucan, the M2fraction was dissolved in 0.5 mol/L of an acetated buffer (pH 4.3),amyloglucosidase (Sigma Chem. Co.) was added thereto, and the whole wasreacted at 60° C. for 30 minutes. After the whole was adjusted to pH4.5, glucoamylase (Wako Pure Chemical Industries) was added thereto, andthe whole was reacted at 60° C. for 30 minutes. The whole was applied togel filtration using Sephadex G-25 to collect a high molecular fraction(carbohydrates:proteins=13:87). The decrease of α-glucan was confirmedby an NMR analysis.

Further, to prepare a fraction with reduced β-glucan, the M2 fractionwas dissolved in 0.5 mol/L of an acetated buffer (pH 4.3), β-glucosidase(Sigma Chem. Co.) was added thereto, and the whole was reacted at 37° C.for 24 hours. The whole was applied to gel filtration using SephadexG-25 to collect a high molecular fraction(carbohydrates:proteins=59:41). The decrease of β-glucan was confirmedby an NMR analysis.

The activity of cells from a mesenterium lymph node was evaluated in amanner similar to that in Example 5.

The results are shown in Table 10. The “control group” in Table 10 meansa control group in which distilled water was administered. A significanttest was carried out with respect to the control group, and “NS” means“Not Significant”.

As apparent from Table 10, when the peptide portion in the M2 fractionwas treated with hydrazine to decompose and remove the peptide portion,the activity clearly disappeared. Further, when the carbohydrate portionin the M2 fraction was treated with amyloglucosidase and glucoamylase toreduce α-glucan, the activity was decreased, but the activity was notaffected by the β-glucosidase treatment.

The results suggested that the active structure of the M2 fraction wasα-glucan and proteins. TABLE 10 MLR-CMC (% to Treatment to M2 fractioncontrol group) Significant test Non-treated 177 ± 32 p < 0.01 Hydrazinetreatment 112 ± 19 NS Amyloglucosidase and 137 ± 27 NS glucoamylasetreatment β-glucosidase treatment 165 ± 25 p < 0.01

Referential Example 1 Preparation of an Adsorption Fraction of anExtract Mixture from Commercially Available Tricholoma matsutake by anAnion Exchange Resin

After 100 g of commercially available Tricholoma matsutake fruit bodiesharvested in Nagano Prefecture was lyophilized to remove water, thefruit bodies were crushed to obtain 15 g of powder.

The extraction and fractionation procedures in Example 1 was repeated,except that the fruit body powder was used instead of the mycelia as astarting material, to obtain a non-adsorption fraction ml and anadsorption fraction m2.

Industrial Applicability

The immune enhancing agent of the present invention can enhance animmune activity in a subject in need of an immune enhancement, and iseffective, for example, in treating or preventing cancer, particularly aprimary tumor or metastatic foci. Further, the agent of the presentinvention for promoting a recovery from stress can promote such arecovery from stress.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. An adsorption fraction of a liquid mixture by an anion exchangeresin, wherein the liquid mixture is obtainable by mixing a hot waterextract of a mycelium of Tricholoma matsutake FERM BP-7304 with analkaline solution extract of a mycelial residue obtained when preparingthe hot water extract, and (a) the content of carbohydrates in theadsorption fraction is 60 to 72% as a glucose equivalent determined by aphenol-sulfuric acid method, and (b) the content of proteins in theadsorption fraction is 28 to 40% as an albumin equivalent determined bya copper-Folin method.
 2. An immune enhancing agent comprising as anactive ingredient the adsorption fraction according to claim
 1. 3. Animmune enhancing composition comprising the adsorption fractionaccording to claim 1 and a pharmaceutically acceptable carrier.
 4. Animmune enhancing health food comprising the adsorption fractionaccording claim 1, alone or, optionally, with one or more foodcomponents.
 5. The immune enhancing health food according to claim 4,wherein the health food is a functional food.
 6. A method for an immuneenhancement, comprising administering to a subject in need thereof theadsorption fraction according to claim 1 in an amount effectivetherefor.
 7. Use of the adsorption fraction according to claim 1 in themanufacture of an immune enhancing composition or health food.
 8. Anagent for treating or preventing metastatic foci, comprising as anactive ingredient the adsorption fraction according to claim
 1. 9. Acomposition for treating or preventing metastatic foci, comprising theadsorption fraction according to claim 1 and a pharmaceuticallyacceptable carrier.
 10. A health food for treating or preventingmetastatic foci, comprising the adsorption fraction according to claim1, alone or, optionally, with one or more food components.
 11. Thehealth food according to claim 10, which is a functional food.
 12. Amethod for treating or preventing metastatic foci, comprisingadministering to a subject in need thereof the adsorption fractionaccording to claim 1 in an amount effective therefor.
 13. Use of theadsorption fraction according to claim 1 in the manufacture of acomposition or health food for treating or preventing metastatic foci.14. An agent for increasing a serum IAP value, comprising as an activeingredient the adsorption fraction according to claim
 1. 15. Acomposition for increasing a serum IAP value, comprising the adsorptionfraction according to claim 1 and a pharmaceutically acceptable carrier.16. A health food for increasing a serum IAP value, comprising theadsorption fraction according to claim 1, alone or, optionally, with oneor more food components.
 17. The health food according to claim 16,which is a functional food.
 18. A method for increasing a serum IAPvalue, comprising administering to a subject in need thereof theadsorption fraction according to claim 1 in an amount effectivetherefor.
 19. Use of the adsorption fraction according to claim 1 in themanufacture of a composition or health food for increasing a serum IAPvalue.
 20. An agent for promoting a recovery from stress, comprising asan active ingredient the adsorption fraction according to claim
 1. 21. Acomposition for promoting a recovery from stress, comprising theadsorption fraction according to claim 1 and a pharmaceuticallyacceptable carrier.
 22. A health food for promoting a recovery fromstress, comprising the adsorption fraction according to claim 1, aloneor, optionally, with one or more food components.
 23. The health foodaccording to claim 22, which is a functional food.
 24. A method forpromoting a recovery from stress, comprising administering to a subjectin need thereof the adsorption fraction according to claim 1 in anamount effective therefor.
 25. Use of the adsorption fraction accordingto claim 1 in the manufacture of a composition or health food forpromoting a recovery from stress.